The present invention relates to a method and an apparatus for recovering separated cultured cells.
Techniques for culturing living organisms and cells are known as basic experimental techniques to assist cytological studies in the fields of medicine, biology, pharmacology, agriculture and the like. Subculture of organisms and cells utilizing such techniques is generally performed according to the following procedures.
A culture solution in a culture container in which cells have been cultured is discharged therefrom, leaving the cultured cells behind. A buffer is poured into the container to rinse the cell surfaces, and the buffer is then also discharged. Subsequently, an enzyme solution containing a proteinase is poured into the container. After the container has been left to stand for 1 minute, the enzyme solution is discharged, and the container is left to stand for a further period of 5 to 10 minutes. Fresh culture solution is then poured into the container and stirred so as to remove therefrom the cultured cells attached to the bottom surface of the container, and to separate them from each other. The culture solution containing separated cells is then poured into other culture containers for further multiplication.
In a series of culture procedures as described above, the steps of dislodging the cells from the bottom of the container by stirring, and of pouring the fresh culture solution into other culture containers, most affect the subsequent growth of the cultured cells. These steps are the most important factors in determining the recovery rate of the cells, the degree of subsequent cell attachment, damage to the cell membranes, and so on. For these reasons, these are the most important steps in subculture techniques.
Removal of the cells from the growing surface of the container is conventionally performed by the following method. First, a lid of a culture container such as a petri dish is opened. The culture solution in the petri dish is repeatedly drawn and discharged with a pipette so as to remove the cultured cells attached to the growing surface or bottom surface of the petri dish, and to separate the cells from each other. When this method is performed manually, the pipette may be freely moved or pivoted so as to remove the cells uniformly. However, when the method is performed automatically with a machine, various problems are encountered. For example, an apparatus is known which draws and discharges the culture solution by moving the distal end of a pipette to and fro. However, with this apparatus, the range of the growing surface from which the cells are removed is limited, resulting in irregular cell removal. In order to prevent irregular cell removal, the petri dish may be rotated or the delivery rate of the pipette may be increased. However, the structure of the apparatus becomes complex with the former measure, while the culture solution may be spilt outside the petri dish with the latter measure. Furthermore, an apparatus for removing the cells with a pipette requires a long time for complete removal. In addition, the removal procedure is performed with the lid of the petri dish open, so that the cultured cells and culture solution are easily subject to contamination.
Another method for removing cultured cells from a growing surface is known in which an impact is repeatedly applied to a culture container in a direction perpendicular to the growing surface. FIG. 1 shows an apparatus for practicing this method. The apparatus has a support frame 100 of an L-shaped cross-section, an annular base 102 mounted on the support frame 100, and a platform 104 mounted on the annular base 102. A pair of solenoids 106 and 108 are mounted on the support frame 100 and below the platform 104, and have plungers 110 and 112, respectively, which project toward the lower surface of the platform 104. The solenoids 106 and 108 are connected to a drive circuit 114. The drive circuit 114 supplies a pulsed drive current to drive these solenoids 106 and 108. A mounting member 116 is fixed on the upper end of the support frame 100. A buffer member such as a coil spring 118 is mounted to the mounting member 116 so as to oppose the platform 104.
A case in which the cultured cells are removed using the apparatus of the construction as described above will now be described. A petri dish 120 as a culture container is prepared. Cultured cells 122 are attached to the bottom surface of the petri dish 120, and a culture solution 124 is held in the petri dish 120. The petri dish 120 is subjected to the following operations: discharge of the used culture solution, pouring and discharge of a buffer, pouring and discharge of an enzyme solution, and pouring of a fresh culture solution. Thereafter, a lid 126 of the petri dish 120 is closed and the petri dish 120 is placed on the platform 104. The lid 126 is pressed downward by the buffer member 118 so that the petri dish 120 is securely held on the platform 104. A pulsed drive current from the drive circuit 114 is alternately supplied to the solenoids 106 and 108. Then, the plungers 110 and 112 alternately strike or impact against the bottom wall of the petri dish 120 through the platform 104. Since the petri dish 120 is elastically held by the buffer member 118, accidental opening of the lid 126 and resultant spillage of the culture solution may be prevented. Slippage of the petri dish 120 from the platform 104 is also prevented. Accordingly, the petri dish 120 is struck a sufficient number of times. The petri dish 120 is repeatedly struck for about 1 minute to completely dislodge all the cultured cells from the growing surface or the bottom surface of the petri dish 120.
However, when the cultured cells are removed by the method and apparatus as described above, more than one solenoid must be used, making the apparatus complicated. Control for alternately energizing the solenoids is difficult. The impacts acting upon the growing surface in the direction perpendicular thereto do not effectively dislodge the cells from the growing surface. For this reason, the cell removal rate largely depends upon the effectiveness of other steps such as enzyme treatment. For example, if the enzyme treatment is insufficient and dissolution of the cytoplasm bonding the cells to the growing surface is insufficient, the cell removal rate is significantly degraded. Furthermore, since the impacts act upon the bottom wall of the petri dish 120 which is relatively weak, damage to the petri dish 120 and the cells contained therein may be easily caused. Although the degree of difficulty in cell removal may vary depending upon the condition of the cells, in particular, the age of the cells, the apparatus fails to allow free selection of the frequency or magnitude of the impacts to be applied.